Stereoselective Syntheses of 3′-Hydroxyamino- and 3′-Methoxyamino-2′,3′-Dideoxynucleosides

Aminonucleosides are used as key motifs in medicinal and bioconjugate chemistry; however, existing strategies toward 3′-hypernucleophilic amine systems do not readily deliver deoxyribo-configured products. We report diastereoselective syntheses of deoxyribo- and deoxyxylo-configured 3′-hydroxyamino- and 3′-methoxyamino-nucelosides from 3′-imine intermediates. The presence or absence of the 5′-hydroxyl-group protection dictates facial selectivity via inter- or intramolecular delivery of hydride from BH3 (borane). Protecting group screening gave one access to previously unknown 3′-methoxyamino-deoxyguanosine derivatives.

A mino-functionalized nucleosides are key fragments for the development of antiviral agents, nucleic acids technologies, and bioconjugates. While the introduction of azafunctionalities at the 5′-position is relatively straightforward because of the limited effect of steric hindrance, 3′functionalization is more challenging. Modified ribo-and deoxyribo-nucleosides with hydroxyamino and methoxyamino groups at their 3′-positions possess antiviral, anti-leukemic, and anti-HIV activities. 1 For example, the growth of L1210 cells was shown to be inhibited by 2′-deoxy-2′-(hydroxyamino) cytidine with an IC 50 of 1.84 μM; however, synthesis was achieved indirectly, via a uridine derivative. 1b Tronchet et al. 2 explored the synthesis of 3′-methoxyamino-and 3′-hydroxyamino-derivatives by stereoselective reduction of 3′-imines. They readily obtained deoxyxylo-configured systems as major or exclusive products across a range of reduction conditions. The deoxyribo-isomers, on the other hand, were usually minor products or absent, where syntheses have only been achieved via indirect, multistep methods. Richert, Szostak, and their coworkers have also exploited the nucleophilicity of amines for chemical primer extension studies; however, they have not taken advantage of the enhanced nucleophilicities of hypernucleophilic amines. 3 Thus, we sought to develop a stereoselective reduction strategy to access deoxyribo-configured 3′hydroxyamino-and 3′-methoxyamino-nucleoside systems directly from 3′-imine intermediates.
Our initial investigations centered on thymidine systems because they do not require nucleobase protection and show reasonable solubility properties. We chose 5′-O-TBDMS-2,3dideoxy-3-N-methoxyimino-thymidine 1 as our starting material, and it was prepared according to reported procedures. 4,2a Tronchet et al. 2a reported the use of NaBH 3 CN to reduce 1, albeit with low levels of conversion; thus, we explored the use of Bu 3 SnH/BF 3 ·Et 2 O, 5 L-selectride, 6 and NaBH 4 ; 7 however, in all cases, we were unable to obtain the desired ribo-configured compound 3 (Scheme 1), and the xyloproduct was formed instead. Sebesta et al. 8 and Matsuda and co-workers 1b successfully synthesized 2′-(alkoxyamino)uridines via the intramolecular nucleophilic substitution upon 2,2′-O-anhydrouridine derivatives. Thus, we attempted nucleophilic substitution at the 3′position of 2,3′-anhydrothymidine with methoxylamine under a range of reaction conditions; however, surprisingly, we only observed a hydrolytic opening of the anhydro-linkage.
Stereoselective reduction of 3′-keto nucleosides to ribonucleosides via intramolecular delivery of hydride, tethered through a free 5′-hydroxyl group, has been reported. 9 Moreover, Matsuda and co-workers 1b reported that 3′-(hydroxyamino) uridine with a ribo-configuration 5a can be obtained from the corresponding 3′-hydroxyiminouridine 4a by treatment with NaBH 4 /AcOH (Scheme 2). Thus, we attempted the reduction of imine 4b under similar conditions; however, poor conversion to 5b was observed (Scheme 2). This result aligns with the findings of Tronchet et al., 2 who used NaBH 3 CN upon 1 under acidic conditions to obtain low levels of the deoxyribomethoxyamino-product 5b as part of a complex mixture that prevented the isolation of pure material.
We then explored the application of the borane− tetrahydrofuran complex for the reduction of 4b, which we expected to show higher reactivity and higher levels of conversion. To our delight, we obtained 3′-methoxyaminothymidine 5b with the desired deoxyribo-configuration exclusively in 72% yield (Scheme 3). We were also able to reduce protected imine 1 with BH 3 ·THF to give deoxyxyloconfigured product 2 in a yield of 70%. We sought to confirm the absolute configurations of the deprotected 3′-methoxyamino-products 5b and 6 by 2D NMR spectroscopy. Unfortunately, the signals arising from the 3′-H [NCH-(OMe)], 4′-H (OCH), and the 5′-H (OCH 2 OTBS) protons were overlapping in the 1 H NMR spectra, thus preventing clear assignments by NOESY correlations. We also attempted similar analyses using the 5′-TBS-protected systems 2 and 3; however, we encountered the same signal overlap problems. Thus, in order to increase the chemical shifts of the 5′-H signals and, to a lesser extent, 4′-H signals, we prepared 5′tosyl derivatives 7 and 8. This strategy allowed us to distinguish and assign each of the proton signals around the sugar rings. The deoxyribo-isomer 7 did not show NOESY correlation between the 3′-and the 1′-protons, whereas correlations were clearly observed for the deoxyxylo-isomer 8.
Additionally, in the case of deoxyribo-isomer 7, NOESY signals were observed between the 3′-proton and thymine nucleobase, along with the expected NOESY correlation between the 4′and the 1′-protons. The xylo-isomer 8 also showed the expected 4′−1′ NOESY correlations.
In order to gain mechanistic insights into the proposed intramolecular hydride delivery via complexation of the boron to the free hydroxyl group at the 5′-position, we carried out 11 B NMR experiments. 10 The 5′-TBS protected thymidine imine 1 and deprotected 3′-methoxyimino thymidine 4b were treated with B(OMe) 3 in THF-d 8 . Starting with the addition of 0.5 equiv of B(OMe) 3 , 11 B NMR spectra were recorded for multiple additions of 0.5 equiv of B(OMe) 3 up to 2.5 equiv. Figure 1 gives evidence for B−N complexation via the imine nitrogen of 5′-TBS-protected 3′-methoxyimino-thymidine 1 via a signal at 19.19 ppm, which persists even after overnight incubation with 2.5 equiv of B(OMe) 3 . In the case of the 5′hydroxy 3′-methoxyimino-thymidine 4b, we observed two distinct signals at 22.98 ppm (RO−B−N) and 19.20 ppm that indicate the complexation of boron with the free hydroxyl group at the 5′-position and B−N complex, respectively (Figure 1). 11 Taken together, these simple experiments support the idea of a critical role for 5′-OH complexation in the reduction of 4b to deliver the deoxyribo-configuration observed in 5b.
On the basis of our promising results with the thymidine system, we applied the same strategies to the adenosine and cytidine systems. Reduction with BH 3 ·THF was successfully performed on 5′-OH-and 5′-OTBS-3′-methoxyimino-2′,3′dideoxycytidine systems 12 to afford deoxyribo-product (9a) and deoxyxylo-product (9b), respectively, in 71% and 68% yields (Figure 2). The 5′-OH-3′-methoxyimino-2′,3′-dideoxyadenosine system 12 afforded the deoxyribomethoxylamine product 10 exclusively, which was derivatized at the 5′-position ( Figure 2) to minimize conformational changes and, thus, confirm configuration (see the Supporting Information). 14, 2b We then moved on to explore the application of our BH 3 · THF reduction strategies toward guanosine systems. Guanosine systems present significant synthetic challenges because of their poor solubility properties. 13 With this in mind, we attempted reductions on the 5′-OTBS-N-isobutyroyl-protected methoxyimino-derivative of deoxyguanosine and the analogous 5′-OH system 12 using BH 3 ·THF. These reactions resulted in the reduction of the imines to the desired deoxyxylo-product (11b) and deoxyribo-product (11a) in 85% and 70% yield, respectively, but the isobutyroyl group was also reduced. Thus, we moved to a N-DMT-protected substrate, which tolerated BH 3 ·THF to yield the deoxyribo-product 12 after TBS protection, as its tosic acid salt in 80% yield upon deprotection of the DMT group (Figure 3). The configurations of the derivatives of all guanosine products were confirmed by NOESY analysis of the 5′-derivatives (see the Supporting Information).
In conclusion, we have developed efficient, direct strategies to obtain deoxyribo-and deoxyxylo-isomers of 3′-methoxyamino-and 3′-hydroxyamino-deoxynucleosides, from common    intermediates, via stereoselective reductions of the corresponding 3′-imino deoxynucleosides using BH 3 ·THF. Our approach has delivered ribo-configured deoxynucleosides in good yields, which are otherwise difficult to obtain. To the best of our knowledge, the ribo-deoxycytidine derivative 9a, deoxyadenosine derivative 10, and ribo-and xylo-deoxyguanosine derivatives 11a−c and 12 containing the 3′-methoxyaminofunctionality are novel compounds.
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Notes
The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS
We are grateful to BBSRC for funding this research through grant number BB/P02145X/1.